The localization of proteins to different cellular and extracellular compartments to carry out various functions is of fundamental importance to all living cells. This project will center on the general secretion pathway (the Sec pathway) Escherichia coli which involves SecA, SecB, SecY, SecE, SecG, SeeD, SecF and YajC. We will continue to combine molecular manipulation and biochemical studies in our well established in vitro systems with E. coli inverted membrane vesicles. In addition, we will extend the complementary electrophysiological and physical approaches that are newly developed for further studies. Building on the recent unexpected findings, though if somewhat against the current dogma, that some SecA integrates into membranes and does not cycle on and off membranes during translocation, we will test the hypothesis that in addition to catalyzing ATP hydrolysis, certain domains of SecA play an important structural role in the translocation machinery, forming part of the protein-conducting channels. This hypothesis is gaining further support with the recent findings that ionic current activity of the protein-conducting channels can be observed in the absence of SecYEG complex, and that SecA alone forms ring-like and dumbbell structures upon interaction with anionic phospholipids. These new exciting findings in the context of how SecA functions in the membranes, as well as the roles of SecYEG in protein translocation will be further explored in this project. The specific aims are: 1.) To further characterize the functions of membrane SecA in the Sec secretion pathway: (a) to determine the function of lipid-specific domains of SecA in the membranes; (b) to determine the role of SecA in electro-current activity of the protein conducting channel; (c) to determine the structures of SecA upon interaction with phospholipids; and 2). To determine the roles of SecYEG and other proteins in protein translocation: (a) to determine the functions of SecYEG; (b) to determine the roles of other proteins; and (c) to test a simplified working model. Characterizing the roles of SecYEG and SecA in the membranes as proposed here is of fundamental significance to understanding the mechanisms of bacterial protein secretion, which has important medical and industrial applications.